ADAR1 exacerbates ischemic brain injury via astrocyte-mediated neuron apoptosis.

Astrocytes affect stroke outcomes by acquiring functionally dominant phenotypes. Understanding molecular mechanisms dictating astrocyte functional status after brain ischemia/reperfusion may reveal new therapeutic strategies. Adenosine deaminase acting on RNA (ADAR1), an RNA editing enzyme, is not normally expressed in astrocytes, but highly induced in astrocytes in ischemic stroke lesions. The expression of ADAR1 steeply increased from day 1 to day 7 after middle cerebral artery occlusion (MCAO) for 1 h followed by reperfusion. ADAR1 deficiency markedly ameliorated the volume of the cerebral infarction and neurological deficits as shown by the rotarod and cylinder tests, which was due to the reduction of the numbers of activated astrocytes and microglia. Surprisingly, ADAR1 was mainly expressed in astrocytes while only marginally in microglia. In primary cultured astrocytes, ADAR1 promoted astrocyte proliferation via phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Furthermore, ADAR1 deficiency inhibited brain cell apoptosis in mice with MCAO as well as in activated astrocyte-conditioned medium-induced neurons in vitro. It appeared that ADAR1 induces neuron apoptosis by secretion of IL-1ß, IL-6 and TNF-α from astrocytes through the production of reactive oxygen species. These results indicated that ADAR1 is a novel regulator promoting the proliferation of the activated astrocytes following ischemic stroke, which produce various inflammatory cytokines, leading to neuron apoptosis and worsened ischemic stroke outcome.

Surfactant protein A promotes atherosclerosis through mediating macrophage foam cell formation.

BACKGROUND: Atherosclerosis is a progressive inflammatory disease where macrophage foam cells play a central role in the pathogenesis. Surfactant protein A (SPA) is a lipid-associating protein involved with regulating macrophage function in various inflammatory diseases. However, the role of SPA in atherosclerosis and macrophage foam cell formation has not been investigated. METHODS: Primary resident peritoneal macrophages were extracted from wildtype (WT) and SPA deficient (SPA -/- ) mice to determine the functional effects of SPA in macrophage foam cell formation. SPA expression was assessed in healthy vessels and atherosclerotic aortic tissue from the human coronary artery and WT or apolipoprotein e-deficient (ApoE -/- ) mice brachiocephalic arteries fed high fat diets (HFD) for 4 weeks. Hypercholesteremic WT and SPA -/- mice fed a HFD for 6 weeks were investigated for atherosclerotic lesions in vivo . RESULTS: In vitro experiments revealed that global SPA deficiency reduced intracellular cholesterol accumulation and macrophage foam cell formation. Mechanistically, SPA -/- dramatically decreased CD36 cellular and mRNA expression. SPA expression was increased in atherosclerotic lesions in humans and ApoE -/- mice. In vivo SPA deficiency attenuated atherosclerosis and reduced the number of lesion-associated macrophage foam cells. CONCLUSIONS: Our results elucidate that SPA is a novel factor for atherosclerosis development. SPA enhances macrophage foam cell formation and atherosclerosis through increasing scavenger receptor cluster of differentiation antigen 36 (CD36) expression.

Blood serum analysis: A modified sandwich enzyme-linked immunosorbent assay protocol.

Blood serum analysis is a versatile tool used in diagnostics, in vivo research, and clinical studies. Enzyme-linked immunosorbent assay (ELISA) is a common method used to analyze blood serum cytokine levels; however, commercial kits are costly and not always available for novel or uncommon targets. Here we present a modified ELISA protocol that, once standardized, can be used to measure blood serum levels of any target and minimize the expense of commercial kits. Additionally, this method can be used for novel or unique targets for which commercial options are unavailable. Ultimately, the modified ELISA method is an efficient, cost-effective method of supplementing clinical and in vivo studies with consistently reliable serum cytokine measurements.